Multimodal immobilization of second-instar Drosophila melanogaster larvae using PF-127 hydrogel and diethyl ether for calcium imaging

Motion artifacts remain a barrier to in vivo calcium imaging in Drosophila melanogaster larvae. Here, we evaluate a multimodal immobilization approach that combines a Pluronic F-127 (PF-127) hydrogel with brief diethyl ether vapor exposure (5 minutes, 25{degrees}C) and compare it against hydrogel-only immobilization using custom MATLAB-based analysis software that performs NoRMCorre rigid motion correction. In wide-field GFP recordings at 1 Hz over approximately 60 minutes (N = 15 per group), the multimodal condition significantly reduced motion across all three core metrics after FDR correction (all q < 0.001), with large effect sizes for mean speed (Hedges g = -1.18) and median step size (g = -1.36). In a secondary analysis of the first 30 minutes, uniformly large effect sizes (|g| = 1.10-1.51) were observed, consistent with stronger initial chemical immobilization that partially wanes over the recording period. We implemented a dual-flag quality control system that distinguishes motion data reliability from ROI detection eligibility. Control calcium recordings (33.33 Hz, [~]5 minutes; N = 23) yielded 368 ROIs with a mean SNR 30.4 {+/-} 16.9 and an event rate of 0.228 {+/-} 0.113 Hz. Experimental recordings (N = 21) yielded 295 ROIs with SNR 18.0 {+/-} 10.6 and event rate 0.309 {+/-} 0.188 Hz. SNR was higher in controls (Cliffs{delta} = 0.50, p < 0.001), while event rate was modestly higher in the experimental group at the ROI level ({delta} = -0.22, p < 0.001), though this difference did not reach significance at the sample level, suggesting altered but not suppressed calcium dynamics. These results support a practical, accessible immobilization workflow for larval calcium imaging. HighlightsO_LIBrief ether + hydrogel approach reduces larval motion 85-91% vs. hydrogel alone C_LIO_LIDual-flag QC system separates motion reliability from calcium ROI eligibility C_LIO_LICalcium event rates not suppressed under multimodal immobilization C_LIO_LIComplete MATLAB pipeline for motion analysis and calcium imaging provided C_LIO_LIAccessible protocol requires only standard laboratory supplies C_LI